1. Field of the Invention
The present invention relates to assays for biomarkers useful in the diagnosis of fibrosis disease and prognosis of its development, including biomarkers indicative of the risk of developing fibrosis after a chronic injury.
In particular, according to the present invention, biomarkers relating to degradation fragments of Collagen type I, III, IV, V, and VI, elastin, C-reactive protein, and proteoglycans including Biglycan, Decorin, Versican, and Perlecan are found to be useful.
2. Description of Related Art
Fibrotic diseases (including those listed in Table 1) are a leading cause of morbidity and mortality, e.g. cirrhosis with 800,000 death per year worldwide1.
TABLE 1Different fibrotic diseases2TissueExamples of CausesLiverViral hepatitisSchistosomiasisSteatohepatitis (Alcoholic or non-alcoholic)LungIdiopathic pulmonary fibrosis (IPF)Systemic sclerosis (Scleroderma)KidneyNephrogenic systemic fibrosis (NSF)DiabetesUntreated hypertensionHeartHeart attackHypertensionAtherosclerosisRestenosisEyeMacular degeneration, retinal and vitreal retinopathySkinSystemic sclerosis and scleroderma, keloids, hypertrophic scars, burns,genetic factorsNFSPancreasAutoimmune/hereditary causesIntestineCrohn's disease/inflammatory bowl diseaseBrainAlzheimer's disease, AIDSBone marrowCancer, ageingMulti-organSurgical complications, chemotherapeutic drug-induced fibrosis, radiation-fibrosisinduced fibrosis, mechanical injuries
A ‘fibrotic disease’ is any disease giving rise to fibrosis, whether as a main or a secondary symptom.
Fibrosis is the end result of chronic inflammatory reactions induced by a variety of stimuli including persistent infections, autoimmune reactions, allergic responses, chemical insults, radiation, and tissue injury. Fibrosis is characterized by the accumulation and reorganization of the extracellular matrix (ECM). Despite having obvious etiological and clinical distinctions, most chronic fibrotic disorders have in common a persistent irritant that sustains the production of growth factors, proteolytic enzymes, angiogenic factors, and fibrogenic cytokines, which together stimulate the deposition of connective tissue elements, especially collagens and proteoglycans, which progressively remodel and destroy normal tissue architecture 3, 4. Despite its enormous impact on human health, there are currently no approved treatments that directly target the mechanisms of fibrosis 5.
The key cellular mediator of fibrosis is the myofibroblast, which when activated serves as the primary collagen-producing cell.
Extracellular Matrix (ECM)
Fibrogenesis is a dynamic process involving complex cellular and molecular mechanisms that usually originates from tissue injury 6. Fibrogenesis is the result of an imbalance in normal ECM regulation that alters the concentration of macromolecules leading to increased tissue size and density, with progressively impaired function. These macromolecules are mainly fibrous proteins with structural and adhesive functions, such as collagens and proteoglycans.
Collagen
Collagens are widely distributed in the human body, i.e. ˜30% of the protein mass in the human body is composed of collagens. Collagens are responsible for the structural integrity of the ECM of most connective tissues. The ECM content results from a fine balance between synthesis and degradation tightly controlled through regulation of gene expression and protein secretion, but also through endogenous protease inhibition and protein degradation by metalloproteinases and cysteine proteases 7-9. Table 2 lists the major collagen types with their major tissue distribution.
TABLE 2Major collagen types and their tissue distribution.Collagen typeTissue distributionIMost connective tissuesIICartilage, vitreous humorIIIExtensible connective tissues,e.g. liver, skin, lung, vascular systemIVBasement membranesVTissues containing collagen IVIMost connective tissuesVIISkin, bladder, oral mucosa, umbilical cord, amnionVIIIMany tissues, especially endotheliumXIIIEndothelial cells, skin, eye, heart, skeletal muscleXIVVessel, bone, skin, cartilage, eye, nerve, tendon, uterusXXIVessel, heart, stomach, kidney, skeletal muscle, placenta
Type I collagen is the most abundant collagen and is found in most connective tissues. It is especially important for the structure of bone and skin where the major collagenous components are type I and III collagens 10.
Collagen type I and III are the major components of liver and lung in a 1:1 ratio in healthy tissue. In addition, collagen type IV and VI are found in the basement membranes in most tissues. The most common localization of type V collagen is within the characteristic collagen fibrils, in association with the collagen type I and III 10.
Some collagens have a restricted tissue distribution: for example, type II, which is found almost exclusively in cartilage 11.
During fibrogenesis the net amount of collagens increases12-14. Table 3 shows by way of example the collagen increase during liver fibrosis.
TABLE 3Changes of the composition of collagen from normal to cirrhotic human liver 15.CollagenCollagenDistributionDistributionCollagennormalcirrhoticTimesnormal livercirrhotictypeChainsliver (mg/g)liver (mg/g)increased(%)liver (%)Iα1(I) α2(I)21683742IIIα1(III)2843721IVα1(IV)0.5714918α2(IV)Vα1(V)0.9781718α2(V)α3(V)VIα1(VI)0.010.1100.20.3α2(VI)Elastin
Elastin is a protein present in many connective tissues, primarily those that are elastic. It has a very high content of the amino acids glycine, valine, alanine, and proline, and has a molecular weight of 64 to 66 kDa. It is organised in an irregular or random coil conformation made up of 830 amino acids. Elastin is made by linking many soluble tropoelastin protein molecules, in a reaction catalyzed by lysyl oxidase, to make a massive insoluble, durable cross-linked array.
Elastin serves an important function in arteries as a medium for pressure wave propagation to help blood flow and is particularly abundant in large elastic blood vessels such as the aorta. Elastin is also very important in the lungs, elastic ligaments and the skin.
Despite much efforts devoted to the understanding of elastin synthesis and turnover, neo-epitopes originating from the proteolytic cleavage of this matrix molecules have until now not been associated with disease development in fibrosis.
Vimentin
Vimentin is a member of the intermediate filament family of proteins. Intermediate filaments are an important structural feature of eukaryotic cells. They, along with microtubules and actin microfilaments, make up the cytoskeleton. Although most intermediate filaments are stable structures, in fibroblasts, vimentin exists as a dynamic structure. This filament is used as a marker for mesodermally derived tissues, and as such has been used as an immunohistochemical marker for sarcomas.
Hertig and coworkers (Hertig et al., J Am Soc Nephrol. 2008 August; 19(8):1584-91) investigated if epithelial-to-mesenchymal transition in renal tubular epithelial cells of subjects with chronic allograft nephropathy could predict the progression of fibrosis in the allograft and measured vimentin expression in 83 biopsies from these. They did find an association between elevated vimentin expression and the intestinal fibrosis score at 1 year after surgery.
In another study of hepatic fibrosis, Meriden and colleagues (Meriden et al., Clin Gastro & Hepatol 2010; 8:289-296) found a significant association between vimentin expression (in biopsies obtained at F0 stage) and fibrosis progression, with elevated levels predicting rapid progression of the hepatic fibrosis. Accordingly, we wanted to investigate if circulating fragments of vimentin could serve as sensitive and specific biomarkers of fibrosis.
Proteoglycans
Proteoglycans are a diverse group of macromolecules, which covalently link a variable number of glycosaminoglycan (GAG) side chains to a core protein 16. These GAGs are polymers of disaccharide repeats (e.g. N-acetyl glucosamine or N-acetyl galactosamine), which are acidic (negatively charged) due to hydroxyl, carboxylated and sulfated side groups on the disaccharide units. This makes them highly hydrophilic, thus aiding the diffusion of water and positive ions (e.g. sodium from extracellular fluids) 17. Furthermore, GAGs have the ability to form non-covalent links with for example hyaluronic acid chains to form even larger molecular complexes 16. Table 4 lists the most studied proteoglycans associated with connective tissue.
TABLE 4Proteoglycans of the extracellular matrix of connective tissueGroupProteoglycansOriginFunctionLarge extracellularAggrecan 18Articular cartilageExtracellular matrix stabilityproteoglycans (aggregatingchondrocytes, intervertebral(hyaluronan binding)and hyaluronan-binding)disc, nasal cartilageVersican 19, 20Connective tissue: fibroblast,Cell-cell and cell-matrixkeratinocytes, smooth muscleinteractionscells, mesangial cellsBinding of sugars in Ca—dependent mannerNeurocan 21Nervous tissueBinds to neural cell adhesion moleculesBrevican 22Nervous tissueExtracellular matrix stabilitySmall Leucine-richDecorin 23Connective tissue, cartilage,Binds to and connect collagenproteoglycans (collagen-bonemolecules (matrix stabilizationbinding)and thickness)OrganogenesisBinding of TGFβBiglycans 24Capillary endothelium, skinCell differentiation(keratinocytes), epithelium ofBinds and connect collagenkidneyfibrilsFibromodulinConnective tissue, bone,Regulate orientation of collagen fibers17cartilageLumican 23Cornea, muscle, cartilage,Controls spacing andkidney, lung, intestinethickness of collagen fibersCell-associatedSerglycins 25Widely distributed toHemopoietic cellproteoglycansendothelium - intercellulardifferentiationcompartmentsAdhesion and activation of lymphoid cellsSyndecans 26Widely distributed - often cellBinds collagens, fibronectin,membrane boundthrombospondin, tenascin and bFGFBetaglycan 27Widely distributedTGFβ receptor and signalingPossible reservoir of TGFβBasement membranePerlecan 28All basement membranesSelective barrier forproteoglycansmacromoleculesCell-adhesionC-Reactive Protein
C-reactive protein (CRP) is an acute phase serum protein produced by the liver in response to different clinical conditions such as, inflammation, infection, or trauma29. The production of CRP is induced by cytokines such as IL-6, released from the affected or damaged tissues. The physiological role of CRP is yet unknown and discussions on its pro- or anti-inflammatory actions are ongoing.
Proteases
The imbalance between synthesis and degradation of ECM during fibrogenesis, results from conversion of the low-density subendothelial matrix into matrix rich in interstitial collagens. The increase in collagen and proteoglycans may be due to one or both of (1) a decrease in protein production and (2) impaired protein degradation, and hence less matrix degradation. The decreased protein degradation has recently received increased attention. In the regulation of this process matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) play important roles, as well as other proteases and their inhibitors, such as cystein proteases and the cystatins.
MMPs
MMPs are a large group of endopeptidases, capable of degrading most if not all components of the ECM. Presently, more than 25 MMPs have been found. MMPs are characterized by an active site containing a metal atom, typically zinc, and are secreted as zymogens. Different MMPs are expressed in different tissues. In Table 5 MMPs in the liver are shown.
TABLE 5MMPs in the liver30-32FamilyProteaseSourceSubstrateCollagenasesMMP-1HSCI, II, III, VII, VIII, X, gelatinMMP-8NeutrophilI, II, III, V, VII, X, gelatinMMP-13HSC, MFB, KCI, II, III, VII, X, gelatinStromelysinsMMP-3HSCIII, IV, V, IX, X, XI, gelatin, laminin, fibronectin,proteoglycans, glycoproteins, elastin, pro-MMP-1/13MMP-10HSCIII, IV, V, gelatin, elastin, aggrecanMMP-11HCPAI-1, week activity against matrix proteinsGelatinasesMMP-2HSC, MBFI, II, III, IV, V, VII, X, XI, gelagin, elastin, lamininMMP-9KC, HSC, HCI, II, III, IV, V, VII, X, XI, gelagin, elastin, lamininMMP-7HSCEntactin, gelatin, elastin, fibronectin, vitronectin, laminin,fibrinogenMetalloelastaseMMP-12MacrophagesElastin, gelatins, IV, laminin, fibronectin, entactin, vitronectin,proteoglycan, myelin basic protein, α1-antitripsinMT-MMPsMMP-14HSC, MFB, KCI, II, III, gelatin, fibronectin, vitronectin, laminin, fibrinogen,MMP-15HC, BDECpro-MMP-2, pro-MMP-13Pro-MMP-2, fibronectin, tenascin, laminin, aggrecan, perlecan
TIMPs block MMPs' proteolytic activity by binding in a substrate- and tissue-specific manner to MMP and membrane-type 1 metalloproteinase in a trimolecular complex (Table 6). During fibrosis TIMP levels increase dramatically, and MMP levels increase modestly or remain relatively static (except MMP-2) which in all gives a decrease in degradation of collagens.
TABLE 6TIMPs in the liver31NameSourcesMetalloproteinase inhibitedTIMP-1HSC, MFB, KC, HCPro-MMP-9, MMP-1, MMP-2, MMP-3,MMP-13TIMP-2KC, HSCMT-MMP-1, MT-MMP-2, proMMP-2,MMP-3, MMP-13, MMP-7TIMP-3HCMT-MMP-1, MT-MMP-2, TACE,MMP-13Fibroblast Activation Protein
Fibroblast Activation Protein alpha subunit (FAPa or FAP, alpha) is an integral membrane gelatinase belonging to the serine protease family. FAPa is the alpha subunit and DPP4 (CD26) the beta subunit of a heterodimeric membrane-bound proteinase complex also known as 170 kDa Melanoma Membrane Gelatinase, Integral Membrane Serine Proteinase and Seprase. Some cells make only FAPa homodimers, some only DPP4 homodimers. The monomer is inactive. FAP, alpha is selectively expressed in reactive stromal fibroblasts of epithelial cancers, granulation tissue of healing wounds, and malignant cells of bone and soft tissue sarcomas33. This protein is thought to be involved in the control of fibroblast growth or epithelial-mesenchymal interactions during development, tissue repair, and epithelial carcinogenesis. It has been shown that expression of FAP increase with the stage of fibrosis34, 35.
Fibrosis Biomarkers
A number of biochemical markers have been suggested for fibrotic diseases, although not specific product of the disease. In Table 7 is an example of biochemical markers of liver fibrosis used in clinical trial. In addition there are a lot of examples of biomarkers of other fibrotic diseases12, 36-42.
TABLE 7summarizes some of the known markers of liver fibrosis.Chronic liverBiomarkerParametersdiseaseReferenceOne parameterCRPNASH43HyaluronanHCV44-47IGF-IHCV48LeptinHCV49PIIIPHCV50Several parametersMP3PIIINP, MMP1HCV51, 52Zheng et al indexHA, PIIICP, PIIINP, Laminin, C-IVChronic53hepatitisLebensztjen et alLaminin-2, C-IV, MMP2, MMP9-TIMP1 indexHBV54indexTenascin, hyaluronana, Colalegn VI, TIMP-1HBV55Tsochatzis et alLeptin, adiponectin, resistinHCV, HBC, NASH56indexPatel et al indexHyaluronan, TIMP-1, α2-macroglobulinHCV57TIMP-1, tenascin, collagen IV, PIIINP, MMP2,NASH58laminin, HyaluronanForns-index (76, 77)Age, platelet count, γGT, cholesterolHCV51, 59-62HIV/HCVFibroTest (76, 78)Haptoglobin, α2-macroglobulin, apolipoproteinHCV45, 51, 60, 61, 63-75A1, γGT, bilirubinHIV/HCVNAFLDNAFLD indiabetespatientsActitestFibroTest + ALTHCV65, 76-78APRI (Wai-index)AST, platelet countHIV/HCV45, 51, 60, 61, 64, 66, 79-87HCV NAFLDHepascoreBilirubin, γGT, hyaluronan, α2-macroglobulin, age,HCV51, 61, 64, 66, 88genderHIV/HCVFIB-4Platelet count, AST, ALT, ageHIV/HCV61, 83SHASTAHyaluronan, albumin, ASTHIV/HCV61FibroindexFORN + APRIHCV89Fibrometer testPlatelet count, prothrombin index, AST, α2-HIV/HCV51, 61, 64, 66, 81macroglobulin, hyaluronan, urea, ageHCV NAFLDNFSAAge, hyperglycaemia, body mass index, platelets,NAFLD81albumin, AST/ALTUltrasound + APRIHCV82Metwally et al indexPlatelet count, albumin, AST, history of bloodHCV90transfusion, HBV core antibodyMohamadnejad et alAge, HBV DNA levels, alkaline phosphatase,HCV91indexalbumin, platelet counts, ASTFibroSpect IIHyaluronan, TIMP-1, α2-macroglobulinHCV85, 92, 93StepwiseCombination of APRI and FibrotestHCV94combinationalgorithmsImbert-Bismut indexα2 macroglobulin, AST, ALT γGT, total bilirubin,HCV95albumin, α1 globulin, α2 globulin, β globulin, γglobulin, apolipoprotein A1Nunes et alAge, Platelets, INR, CD4, AST/ALT, Hyaluronan,HCV/HIV96YKL-40, PIIINPHCVFibroscan +++Fibroscan, Fibrotest, APRI,HCV97
U.S. Pat. No. 5,387,504 describes the neo-epitope VDIPEN released by the action of stromelysin at the aggrecan site N341-F342 and an RIA assay employing a monoclonal antibody specific for this neo-epitope. More generally the use of monospecific antibodies specific for fragments of aggrecan, generated by specific stromelysin cleavage are described. Elevations of stromelysin occur in osteoarthritis, rheumatoid arthritis, atherosclerotic lesions, gout, inflammatory bowel disease (IBD), idiopathic pulmonary fibrosis (IPF), certain cancers, joint injuries, and numerous inflammatory diseases. Stromelysin is reported to be elevated in idiopathic pulmonary fibrosis, and it is alleged that the assay can be conducted on blood or other biological fluids to detect stromelysin cleavage products of aggrecan and that quantitation of such fragments can be used diagnostically in respect of IPF as well as other conditions. However, no evidence for this is provided and there have to our knowledge been no subsequent publications validating this prediction. Such RIA assays have been commercially available for many years and no reports of their successful use in diagnosing or monitoring any fibrotic disease have appeared.
U.S. Pat. No. 7,225,080 discloses a method for diagnosis of an inflammatory, a fibrotic or a cancerous disease in a patient by measuring the values of at least four biochemical markers selected from the group consisting of α2-macroglobulin, AST (aspartate aminotransferase), ALT (alanine aminotransferase), GGT (gammaglutamyl transpeptidase), γ-globulin, total bilirubin, albumin, α1-globulin, α2-globulin, haptoglobin, β-globulin, apoA1, IL-10, TGF-β1, apoA2, and apoB in the serum or plasma of said patient, and subsequently combining said values in order to determine the presence of liver fibrosis and/or liver necroinflammatory lesions in said patient. The patent does not teach the quantitative measurement of peptide fragment carrying neo-epitopes generated during fibrotic disease.
U.S. Pat. No. 6,060,255 describes a method for diagnosing the degree of liver fibrosis, comprising the steps of measuring the concentration of type IV collagen high molecular weight form in a sample using an antibody that specifically binds to type IV collagen, and relating the measurement to the degree of liver fibrosis. Again, no use is made of neo-epitopes produced by proteolytic enzymes acting in the body. The sample is actually digested with pepsin, which may obscure the natural pattern of collagen cleavage in the sample.
U.S. Pat. No. 4,628,027 (Gay) discloses the production of antibodies specific for connective tissue proteins and, more particularly, the production of monoclonal antibodies by fused cell hybrids against human collagens and enzymes involved in collagen degradation. The use of monoclonal antibodies against connective tissue proteins to establish the collagen profile of histological, cytological and biological fluid samples is described. However, the patent does not describe the measurement of connective tissue proteins based on the binding of antibodies to neo-epitopes on said connective tissue proteins.
Guañabens N et al, J Bone Miner Res, 1998 98 evaluated the bone turnover markers N-telopeptide of type I collagen (NTX), C-telopeptide of type I collagen (CTX) and N-terminal pro-peptide of collagen type I (PINP) in patients with primary biliary cirrhosis, a disease with increased hepatic fibrosis. The level of NTX, CTX and PINP were elevated in patients compared to controls and correlated with the histological stage of the disease. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope JYDGKGVG⇓(SEQ ID NO2249). The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR⇓(SEQ ID NO2250). These markers are located in telopeptides of collagen type I and not in the internal part (the triple helical part) of collagen type I. The monoclonal antibodies employed for the PINP assay were raised against an internal epitope in the PINP sequence which is not a neo-epitope.
Møller S et al, Gut., 1999 99 demonstrated that the C-terminal cross linked telopeptide of type I collagen (ICTP) was elevated in alcoholic cirrhosis patients compared to controls. The study described showed that a biochemical marker can reflect hepatic fibrosis. The ICTP polyclonal antibody has been raised against trypsin and collagenase cleaved collagen type I. However, the antibodies are not binding to a neo-epitope.
Rosen H N et al, Calcif Tissue Int, 2004 100 assessed the bone turnover markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in women receiving hormone replacement treatment (HRT). In the study it was observed that the bone turnover markers decreased with treatment. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope JYDGKGVG⇓(SEQ ID NO2249). The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR⇓(SEQ ID NO2250). In contrast to the present invention, these antibodies were used for evaluation of bone metabolism and not fibrosis.
Lein M et al, Eur Urol, 2007 101 evaluated the use of the neo-epitope specific bone turnover markers N-telopeptide of type I collagen (NTX) and C-telopeptide of type I collagen (CTX) in prostate cancer patients receiving zoledronic acid. In the study it was observed that the bone turnover markers decreased with treatment. The antibodies employed in the NTX were raised against a cathepsin K cleaved site in the N-terminal of collagen type I and are dependent on the neoepitope JYDGKGVG⇓(SEQ ID NO2249). The antibodies employed in the CTX were raised against a cathepsin K cleaved site in the C-terminal of collagen type I and are dependent on the neoepitope EKAHDGGR⇓(SEQ ID NO2250). In contrast to the present invention, these antibodies were used for evaluation of the bone metabolism during invasion of bone metastases and not fibrosis.
PIIINP has been used in a number of studies to assess the severity of fibrotic disease 102, in patients with skin fibrosis following severe burn trauma 103, for disease progression in noncirrhotic primary biliary cirrhosis 104, in primary biliary cirrhosis and chronic viral hepatitis C 105.
PIIINP and ICTP were measured in patients with fibrosis of the myocardium 106.
Many reports combine a set of biochemical markers to improve the predictive value of the biochemical index. Eleven different serum markers were measured in 205 patients with fibrotic staging from F0 to F4, and the most informative markers were alpha2 macroglobulin, alpha2 globulin (or haptoglobin), gamma globulin, apolipoprotein A1, gamma glutamyltranspeptidase, and total bilirubin 107. An index of these markers had a negative predictive value (100% certainty of absence of F2, F3, or F4) was obtained for scores ranging from zero to 0.10 (12% [41] of all patients), and high positive predictive value (>90% certainty of presence of F2, F3, or F4) for scores ranging from 0.60 to 1.00 (34% [115] of all patients).
However, in none of the above mentioned reports is it suggested that measurements of peptide fragments based on antibodies binding to neo-epitopes as now claimed might be useful for the assessment of patients with fibrotic disease.